Trypanosomatid organisms cause devastating and life-threatening human disease. Several unique biological properties are being pursued as potential new drug targets for much needed chemotherapeutic interventions. While studies on the mitochondrial kinetoplast DNA network are quite advanced, little information is available about nuclear DNA replication mechanisms or the machinery involved in replication initiation, called Origin Recognition Complex (ORC). In contrast to the six-subunit (Orc1-6) Origin Recognition Complex (ORC) found in all other model eukaryotes, the Tritryp genomes predict just a single ORC subunit, Orc1. In this proposal we will investigate function of this core DNA replication initiation protein, identify other protein components that might participate in origin binding, and identify DNA replication origins. Completion of these specific aims will have a major impact on the field, as they will address a critical gap in our understanding of a basic essential process in Trypanosoma brucei, and lay the foundation for a new frontier in trypanosome biology. Functional characterization of T. brucei Orc1 and interacting proteins may allow use to exploit any differences between trypanosomatid and mammalian replication initiation mechanisms to develop new strategies for drug treatment. Studies on T. brucei Orc1 also offer an opportunity to uncover insights into alternative mechanisms of eukaryotic DNA replication that may be applicable to other parasite systems. PUBLIC HEALTH RELEVANCE: This research is highly relevant to public health because trypanosomatids cause serious and fatal disease around the world for which current therapies are woefully inadequate. DNA replication initiation is fundamental for parasite survival, and core components differ substantially from the host. The proposed studies will greatly advance our understanding of alternative mechanisms of eukaryotic DNA replication initiation, and is a promising approach to novel therapeutic intervention in trypanosomatid pathogens.